In this study, RUNX3 expression was analyzed in normal and malignant haematopoiesis and the impact of its dysregulation on myelopoiesis was further determined. We found that RUNX3 was highly expressed in haematopoietic progenitors, with its levels reducing towards granulocytic differentiation. In AML, RUNX3 was overexpressed across all different subtypes except in core binding factor AML where it was downregulated. RUNX3 overexpression in human haematopoietic stem and progenitor cells (HSPC) inhibited myeloid development, particularly granulopoiesis. Further RNA-sequencing studies showed that RUNX3 overexpression downregulates key hematopoietic genes, while upregulating certain lymphoid genes. Overall, this study suggests that increased RUNX3 expression could contribute to the myeloid block characteristic of AML by possibly driving a competing transcriptional program favoring the lymphoid fate. Overall design: To model the effect of RUNX3 overexpression in AML, human CD34+ HSPC were transduced with either a GFP control or RUNX3 GFP recombinant retrovirus. Following infection, cells were sorted by FACS for GFP positivity and RNA was harvested for RNA sequencing. 5 biological replicates were analyzed in total (Control vs RUNX3).